Diagnostic assays are used in clinical diagnosis and health science research to detect and/or quantify the presence and/or amount of biological antigens, cell abnormalities, disease states, and disease-associated pathogens present in a host organism or biological sample. Exemplary disease-associated pathogens include parasites, fungi, bacteria, and viruses. When a diagnostic assay permits quantification, practitioners can calculate the extent of infection or disease and determine the state of a disease over time. Diagnostic assays can detect, for example, chemicals, proteins, polysaccharides, nucleic acids, biopolymers, cells, or tissue of interest. A variety of assays may be employed to detect and/or qualify these diagnostic indicators.
To detect a targeted nucleic acid sequence, a probe having a nucleotide base sequence that is substantially complementary to the targeted sequence or its amplicon can be used. Under selective assay conditions, the probe can hybridize to the targeted sequence or its amplicon in a manner permitting a practitioner to detect the presence of the targeted sequence in a sample. Probes may include, for example, a detectable label such as a radiolabel, a fluorophore or fluorescent dye, biotin, an enzyme or a chemiluminescent compound. The probe can hybridize to the targeted sequence or its amplicon such that a signal indicating the presence of the targeted sequence in a sample can be detected, and the strength of the signal can be proportional to the amount of the target sequence or its amplicon that is present. By periodically measuring, during the amplification process, a signal indicative of the presence of amplicon, the growth of amplicon over time can be detected. Based on the data collected during this “real-time” monitoring of the amplification process, the amount of the target nucleic acid sequence that was originally in the sample can be ascertained.
To detect different nucleic acid sequences of interest in a single assay, different probes configured to hybridize to different nucleic acid sequences and to emit detectably different signals can be used. For example, different probes configured to hybridize to different target nucleic acid sequences can be formulated with fluorophores that fluoresce at a known wavelength (i.e., color) when exposed to excitation light of a known excitation wavelength. Assays for detecting different target nucleic acid sequences can be performed in parallel by alternately exposing the sample to different excitation wavelengths and detecting the level of fluorescence at the wavelength of interest corresponding to the probe for each target nucleic acid sequence during the real-time monitoring process.
Parallel processing can be performed using different signal detectors configured to periodically measure signal emissions during the amplification process, and with different signal detectors configured to generate excitation signals of different wavelengths and to measure emission signals of different wavelengths. Exemplary signal detectors include fluorometers. One embodiment of an automated nucleic acid assay instrument is configured to process numerous samples carried in multiple receptacles, and each fluorometer is configured to acquire fluorometric readings from the receptacles as they are indexed past the fluorometer, for example, once every 2 seconds. Thus, 1800 times for each hour of operation of the instrument, each fluorometer can generate an excitation signal that is directed at the sample receptacle, and each fluorometer can measure the emission signal emitted by the contents of the receptacle and can generate an electrical signal that is proportional to the intensity of the emission signal. A fluorometer malfunction (e.g., device failure or deteriorated performance) during operation of the instrument will cause errors in the fluorometric readings generated by that fluorometer and thereby cause errors in the diagnostic results. Such malfunctions can be due to mechanical or electrical failures that occur during operation of the fluorometer. While the operation of the fluorometers can be checked during routine maintenance of the instrument, such opportunities for testing are rare because the testing can only be performed when the instrument is shut down. But the instrument can be operated continuously for extended periods of time for maximum throughput. Therefore, repeatedly shutting the instrument down to perform fluorometer functionality testing can be impractical and costly. Accordingly, a need exists for means and methodologies for periodically confirming the proper functionality of the signal detector, for example, a fluorometer, during the normal operation of the nucleic acid diagnostic instrument—while the assay is being performed.